Kir4.1 channels regulate swelling of astroglial processes in experimental spinal cord edema

In glial cells, inwardly rectifying K⁺ channels (Kir) control extracellular [K⁺]_o homeostasis by uptake of K⁺ from the extracellular space and release of K⁺ into the microvasculature. Kir channels were also recently implicated in K⁺-associated water influx and cell swelling. We studied the time-dependent expression and functional implication of the glial Kir4.1 channel for astroglial swelling in a spinal cord edema model. In this CNS region, Kir4.1 is expressed on astrocytes from the second postnatal week on and co-localizes with aquaporin 4 (AQP4). Swelling of individual astrocytes in response to osmotic stress and to pharmacological Kir blockade were analyzed by time-lapse-two-photon laser-scanning microscopy in situ . Application of 30% hypotonic solution induced astroglial soma swelling whereas no swelling was observed on astroglial processes or endfeet. Co-application of hypotonic solution and Ba²⁺, a Kir channel blocker, induced prominent swelling of astroglial processes. In Kir4.1−/− mice, however, somatic as well as process swelling was observed upon application of 30% hypotonic solutions. No additional effect was provoked upon co-application with Ba²⁺. Our experiments show that Kir channels prevent glial process swelling under osmotic stress. The underlying Kir channel subunit that controls glial process swelling is Kir4.1, whereas changes of the glial soma are not substantially related to Kir4.1.     

Kir5.1 underlies long-lived subconductance levels in heteromeric Kir4.1/Kir5.1 channels from Xenopus tropicalis

The inwardly-rectifying potassium channel subunit Kir5.1 selectively co-assembles with members of the Kir4.0 subfamily to form novel pH-sensitive heteromeric channels with unique single channel properties. In this study, we have cloned orthologs of Kir4.1 and Kir5.1 from the genome of the amphibian, Xenopus tropicalis (Xt). Heteromeric XtKir4.1/XtKir5.1 channels exhibit similar macroscopic current properties to rat Kir4.1/Kir5.1 with a faster time-dependent rate of activation. However, single channel analysis of heteromeric XtKir4.1/XtKir5.1 channels reveals that they have markedly different long-lived, multi-level subconductance states. Furthermore, we demonstrate that the XtKir5.1 subunit is responsible for these prominent subconductance levels. These results are consistent with a model in which the slow transitions between sublevel states represent the movement of individual subunits. These novel channels now provide an excellent model system to determine the structural basis of subconductance levels and contribution of heteromeric pore architecture to this process.     

Expression of Kir2.1 channels in astrocytes under pathophysiological conditions

Astrocyte ion channels participate in ionic homeostasis in the brain. Inward rectifying potassium channels (Kir channels) in astrocytes have been particularly implicated in K(+) homeostasis because of their high open probability at resting potential and their increased conductance at high concentrations of extracellular K(+). We examined the expression of the Kir2.1 subunit, one of the Kir channel subunits, in the mouse brain by immunohistochemistry. Kir2.1 channels were widely distributed throughout the brain, with high expression in the olfactory bulb and the cerebellum. Interestingly, they were abundantly expressed in astrocytes of the olfactory bulb, while astrocytes in other brain regions including the hippocampus did not show any detectable expression. However, Kir2.1 channel-expressing cells were dramatically increased in the hippocampus by kainic acid-induced seizure and the cells were glial fibrillary acidic protein (GFAP)-positive, which confirms that astrocytes in the hippocampus express Kir2.1 channels under pathological conditions. Our results imply that Kir2.1 channels in astrocyte may be involved in buffering K(+) against accumulated extracellular K(+) caused by neuronal hyperexcitability under phathophysiological conditions.     

The potassium channel Kir4.1 associates with the dystrophin-glycoprotein complex via alpha-syntrophin in glia

One of the major physiological roles of potassium channels in glial cells is to promote "potassium spatial buffering" in the central nervous system, a process necessary to maintain an optimal potassium concentration in the extracellular environment. This process requires the precise distribution of potassium channels accumulated at high density in discrete subdomains of glial cell membranes. To obtain a better understanding of how glial cells selectively target potassium channels to discrete membrane subdomains, we addressed the question of whether the glial inwardly rectifying potassium channel Kir4.1 associates with the dystrophin-glycoprotein complex (DGC). Immunoprecipitation experiments revealed that Kir4.1 is associated with the DGC in mouse brain and cultured cortical astrocytes. In vitro immunoprecipitation and pull-down assays demonstrated that Kir4.1 can bind directly to alpha-syntrophin, requiring the presence of the last three amino acids of the channel (SNV), a consensus PDZ domain-binding motif. Furthermore, Kir4.1 failed to associate with the DGC in brains from alpha-syntrophin knockout mice. These results suggest that Kir4.1 is localized in glial cells by its association with the DGC through a PDZ domain-mediated interaction with alpha-syntrophin and suggest an important role for the DGC in central nervous system physiology.     

Kir channels in the CNS: emerging new roles and implications for neurological diseases

Inwardly rectifying potassium (Kir) channels have long been regarded as transmembrane proteins that regulate the membrane potential of neurons and that are responsible for [K(+)] siphoning in glial cells. The subunit diversity within the Kir channel family is growing rapidly and this is reflected in the multitude of roles that Kir channels play in the central nervous system (CNS). Kir channels are known to control cell differentiation, modify CNS hormone secretion, modulate neurotransmitter release in the nigrostriatal system, may act as hypoxia-sensors and regulate cerebral artery dilatation. The increasing availability of genetic mouse models that express inactive Kir channel subunits has opened new insights into their role in developing and adult mammalian tissues and during the course of CNS disorders. New aspects with respect to the role of Kir channels during CNS cell differentiation and neurogenesis are also emerging. Dysfunction of Kir channels in animal models can lead to severe phenotypes ranging from early postnatal death to an increased susceptibility to develop epileptic seizures. In this review, we summarize the in vivo data that demonstrate the role of Kir channels in regulating morphogenetic events, such as the proliferation, differentiation and survival of neurons and glial cells. We describe the way in which the gating of Kir channel subunits plays an important role in polygenic CNS diseases, such as white matter disease, epilepsy and Parkinson's disease.     

Polyamine permeation and rectification of Kir4.1 channels

Inward rectifier K(+) (Kir) channels are expressed in multiple neuronal and glial cells. Recent studies have equated certain properties of exogenously expressed Kir4.1 channels with those of native K(+) currents in brain cells, as well as demonstrating the expression of Kir4.1 subunits in these tissues. There are nagging problems however with assigning native currents to Kir4.1 channels. One major concern is that in many native tissues, the putatively correlated currents show much weaker rectification than typically reported for cloned Kir4.1 channels. We have now examined the polyamine-dependence of Kir4.1 channels expressed at high density in Cosm6 cells, using inside-out membrane patches. The experiments reveal a complex and variable rectification that can help explain the variability reported for candidate Kir4.1 currents in native cells. Most importantly, rectification seems to be incomplete, even at high polyamine concentrations. In excised membrane patches, with high levels of expression, and high concentrations of spermine, there is approximately 15% residual conductance that is insensitive to spermine. From a biophysical perspective, this is a striking finding, and indicates either that a bound spermine fails to completely block permeation or that significant spermine permeation (i.e. 'punchthrough') is occurring. To examine this further, we have examined block by philanthotoxin (PhTx, essentially spermine with a bulky tail). PhTx block, while less potent, is more complete than spermine block. This leads us to propose that spermine 'punchthrough' may be significant in Kir4 channels, and that this may be a major contributor to the weak rectification observed under physiological conditions.     

Conduction through the inward rectifier potassium channel, Kir2.1, is increased by negatively charged extracellular residues

Ion channel conductance can be influenced by electrostatic effects originating from fixed "surface" charges that are remote from the selectivity filter. To explore whether surface charges contribute to the conductance properties of Kir2.1 channels, unitary conductance was measured in cell-attached recordings of Chinese hamster ovary (CHO) cells transfected with Kir2.1 channels over a range of K+ activities (4.6-293.5 mM) using single-channel measurements as well as nonstationary fluctuation analysis for low K+ activities. K+ ion concentrations were shown to equilibrate across the cell membrane in our studies using the voltage-sensitive dye DiBAC4(5). The dependence of gamma on the K+ activity (a(K)) was fit well by a modified Langmuir binding isotherm, with a nonzero intercept as a(K) approaches 0 mM, suggesting electrostatic surface charge effects. Following the addition of 100 mM N-methyl-D-glucamine (NMG+), a nonpermeant, nonblocking cation or following pretreatment with 50 mM trimethyloxonium (TMO), a carboxylic acid esterifying agent, the gamma-a(K) relationship did not show nonzero intercepts, suggesting the presence of surface charges formed by glutamate or aspartate residues. Consistent with surface charges in Kir2.1 channels, the rates of current decay induced by Ba2+ block were slowed with the addition of NMG or TMO. Using a molecular model of Kir2.1 channels, three candidate negatively charged residues were identified near the extracellular mouth of the pore and mutated to cysteine (E125C, D152C, and E153C). E153C channels, but not E125C or D152C channels, showed hyperbolic gamma-a(K) relationships going through the origin. Moreover, the addition of MTSES to restore the negative charges in E53C channels reestablished wild-type conductance properties. Our results demonstrate that E153 contributes to the conductance properties of Kir2.1 channels by acting as a surface charge.     

Identification of a heteromeric interaction that influences the rectification,gating, and pH sensitivity of Kir4.1/Kir5.1 potassium channels

Heteromultimerization between different potassium channel subunits can generate channels with novel functional properties and thus contributes to the rich functional diversity of this gene family. The inwardly rectifying potassium channel subunit Kir5.1 exhibits highly selective heteromultimerization with Kir4.1 to generate heteromeric Kir4.1/Kir5.1 channels with unique rectification and kinetic properties. These novel channels are also inhibited by intracellular pH within the physiological range and are thought to play a key role in linking K+ and H+ homeostasis by the kidney. However, the mechanisms that control heteromeric K+ channel assembly and the structural elements that generate their unique functional properties are poorly understood. In this study we identify residues at an intersubunit interface between the cytoplasmic domains of Kir5.1 and Kir4.1 that influence the novel rectification and gating properties of heteromeric Kir4.1/Kir5.1 channels and that also contribute to their pH sensitivity. Furthermore, this interaction presents a structural mechanism for the functional coupling of these properties and explains how specific heteromeric interactions can contribute to the novel functional properties observed in heteromeric Kir channels. The highly conserved nature of this structural association between Kir subunits also has implications for understanding the general mechanisms of Kir channel gating and their regulation by intracellular pH.     

Interactions between GABA-B1 receptors and Kir 3 inwardly rectifying potassium channels

γ-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the mammalian brain. It acts via both ionotropic GABA-A and metabotropic GABA-B receptors. We evaluated the interaction of receptors with members of the inwardly rectifying potassium (Kir 3) channel family, which also play an important role in neuronal transmission and membrane excitability. These channels are functionally regulated by GABA-B receptors. Possible physical interactions between GABA-B receptor and Kir 3 channels expressed in HEK cells were evaluated using Bioluminescence Resonance Energy Transfer (BRET) experiments, co-immunoprecipitation and confocal microscopy. Our data indicate that Kir 3 channels and Gβγ subunits can interact with the GABA-B₁ subunits independently of the GABA-B₂ subunit or Kir 3.4 which are ultimately responsible for their targetting to the cell surface. Thus signalling complexes containing GABA-B receptors, G proteins and Kir channels are formed shortly after biosynthesis most likely in the endoplasmic reticulum.     

Differential assembly of inwardly rectifying K+ channel subunits, Kir4.1 and Kir5.1, in brain astrocytes

The inwardly rectifying K+ channel subunit Kir5.1 is expressed abundantly in the brain, but its precise distribution and function are still largely unknown. Because Kir5.1 is co-expressed with Kir4.1 in retinal glial Muller cells, we have compared the biochemical and immunological properties of Kir5.1 and Kir4.1 in the mouse brain. Immunoprecipitation experiments suggested that brain expressed at least two subsets of Kir channels, heteromeric Kir4.1/5.1 and homomeric Kir4.1. Immunolabeling using specific antibodies showed that channels comprising Kir4.1 and Kir5.1 subunits were assembled in a region-specific fashion. Heteromeric Kir4.1/5.1 was identified in the neocortex and in the glomeruli of the olfactory bulb. Homomeric Kir4.1 was confined to the hippocampus and the thalamus. Homomeric Kir5.1 was not identified. Kir4.1/5.1 and Kir4.1 expression appeared to occur only in astrocytes, specifically in the membrane domains facing the pia mater and blood vessels or in the processes surrounding synapses. Both Kir4.1/5.1 and Kir4.1 could be associated with PDZ domain-containing syntrophins, which might be involved in the subcellular targeting of these astrocyte Kir channels. Because heteromeric Kir4.1/5.1 and homomeric Kir4.1 have distinct ion channel properties (Tanemoto, M., Kittaka, N., Inanobe, A., and Kurachi, Y. (2000) J. Physiol. (Lond.) 525, 587-592 and Tucker, S. J., Imbrici, P., Salvatore, L., D'Adamo, M. C., and Pessia, M. (2000) J. Biol. Chem. 275, 16404-16407), it is plausible that these channels play differential physiological roles in the K+ -buffering action of brain astrocytes in a region-specific manner.     

Variable loss of Kir4.1 channel function in SeSAME syndrome mutations

SeSAME syndrome is a complex disease characterized by seizures, sensorineural deafness, ataxia, mental retardation and electrolyte imbalance. Mutations in the inwardly rectifying potassium channel Kir4.1 (KCNJ10 gene) have been linked to this condition. Kir4.1 channels are weakly rectifying channels expressed in glia, kidney, cochlea and possibly other tissues. We determined the electrophysiological properties of SeSAME mutant channels after expression in transfected mammalian cells. We found that a majority of mutations (R297C, C140R, R199X, T164I) resulted in complete loss of Kir4.1 channel function while two mutations (R65P and A167V) produced partial loss of function. All mutant channels were rescued upon co-transfection of wild-type Kir4.1 but not Kir5.1 channels. Cell-surface biotinylation assays indicate significant plasma membrane expression of all mutant channels with exception of the non-sense mutant R199X. These results indicate the differential loss of Kir channel function among SeSAME syndrome mutations.     

Differential expression and distribution of Kir5.1 and Kir4.1 inwardly rectifying K+ channels in retina

Kir5.1 is an inwardly rectifying K⁺ channel subunit whose functional role has not been fully elucidated. Expression and distribution of Kir5.1 in retina were examined with a specific polyclonal antibody. Kir5.1 immunoreactivity was detected in glial Müller cells and in some retinal neurons. In the Kir5.1-positive neurons the expression of glutamic acid decarboxylase (GAD₆₅) was detected, suggesting that they may be GABAergic-amacrine cells. In Müller cells, spots of Kir5.1 immunoreactivity distributed diffusely at the cell body and in the distal portions, where Kir4.1 immunoreactivity largely overlapped. In addition, Kir4.1 immunoreactivity without Kir5.1 was strongly concentrated at the endfoot of Müller cells facing the vitreous surface or in the processes surrounding vessels. The immunoprecipitant obtained from retina with anti-Kir4.1 antibody contained Kir5.1. These results suggest that heterotetrameric Kir4.1/Kir5.1 channels may exist in the cell body and distal portion of Müller cells, whereas homomeric Kir4.1 channels are clustered in the endfeet and surrounding vessels. It is possible that homomeric Kir4.1 and heteromeric Kir4.1/Kir5.1 channels play different functional roles in the K⁺-buffering action of Müller cells. inwardly rectifying potassium channel; heteromerization; glial Müller cells; amacrine cells; potassium siphoning     

Control of pH and PIP2 gating in heteromeric Kir4.1/Kir5.1 channels by H-Bonding at the helix-bundle crossing

Inhibition by intracellular H(+) (pH gating) and activation by phosphoinositides such as PIP(2) (PIP(2)-gating) are key regulatory mechanisms in the physiology of inwardly-rectifying potassium (Kir) channels. Our recent findings suggest that PIP(2) gating and pH gating are controlled by an intra-subunit H-bond at the helix-bundle crossing between a lysine in TM1 and a backbone carbonyl group in TM2. This interaction only occurs in the closed state and channel opening requires this H-bond to be broken, thereby influencing the kinetics of PIP(2) and pH gating in Kir channels. In this addendum, we explore the role of H-bonding in heteromeric Kir4.1/Kir5.1 channels. Kir5.1 subunits do not possess a TM1 lysine. However, homology modelling and molecular dynamics simulations demonstrate that the TM1 lysine in Kir4.1 is capable of H-bonding at the helix-bundle crossing. Consistent with this, the rates of pH and PIP2 gating in Kir4.1/Kir5.1 channels (two H-bonds) were intermediate between those of wild-type homomeric Kir4.1 (four H-bonds) and Kir4.1(K67M) channels (no H-bonds) suggesting that the number of H-bonds in the tetrameric channel complex determines the gating kinetics. Furthermore, in heteromeric Kir4.1(K67M)/Kir5.1 channels, where the two remaining H-bonds are disrupted, we found that the gating kinetics were similar to Kir4.1(K67M) homomeric channels despite the fact that these two channels differ considerably in their PIP(2) affinities. This indicates that Kir channel PIP(2) affinity has little impact on either the PIP(2) or pH gating kinetics.     

Molecular Mechanisms of EAST/SeSAME Syndrome Mutations in Kir4.1 (KCNJ10)

Inwardly rectifying potassium channel Kir4.1 is critical for glial function, control of neuronal excitability, and systemic K⁺ homeostasis. Novel mutations in Kir4.1 have been associated with EAST/SeSAME syndrome, characterized by mental retardation, ataxia, seizures, hearing loss, and renal salt waste. Patients are homozygous for R65P, G77R, C140R or T164I; or compound heterozygous for A167V/R297C or R65P/R199Stop, a deletion of the C-terminal half of the protein. We investigated the functional significance of these mutations by radiotracer efflux and inside-out membrane patch clamping in COSm6 cells expressing homomeric Kir4.1 or heteromeric Kir4.1/Kir5.1 channels. All of the mutations compromised channel function, but the underlying mechanisms were different. R65P, T164I, and R297C caused an alkaline shift in pH sensitivity, indicating that these positions are crucial for pH sensing and pore gating. In R297C, this was due to disruption of intersubunit salt bridge Glu²⁸⁸–Arg²⁹⁷. C140R breaks the Cys¹⁰⁸–Cys¹⁴⁰ disulfide bond essential for protein folding and function. A167V did not affect channel properties but may contribute to decreased surface expression in A167V/R297C. In G77R, introduction of a positive charge within the bilayer may affect channel structure or gating. R199Stop led to a dramatic decrease in surface expression, but channel activity was restored by co-expression with intact subunits, suggesting remarkable tolerance for truncation of the cytoplasmic domain. These results provide an explanation for the molecular defects that underlie the EAST/SeSAME syndrome.     

Control of pH and PIP2 Gating in Heteromeric Kir4.1/Kir5.1 Channels by H-Bonding at the Helix-Bundle Crossing

Inhibition by intracellular H⁺ (pH gating) and activation by phosphoinositides such as PIP₂ (PIP₂ gating) are key regulatory mechanisms in the physiology of inwardly-rectifying potassium (Kir) channels. Our recent findings suggest that PIP₂ gating and pH gating are controlled by an intrasubunit H-bond at the helix-bundle crossing between a lysine in TM1 and a backbone carbonyl group in TM2. This interaction only occurs in the closed state and channel opening requires this H-bond to be broken, thereby influencing the kinetics of PIP₂- and pH-gating in Kir channels. In this addendum, we explore the role of H-bonding in heteromeric Kir4.1/Kir5.1 channels. Kir5.1 subunits do not possess a TM1 lysine. However, homology modelling and molecular dynamics simulations demonstrate that the TM1 lysine in Kir4.1 is capable of H-bonding at the helix-bundle crossing. Consistent with this, the rates of pH and PIP₂ gating in Kir4.1/Kir5.1 channels (two H-bonds) were intermediate between those of wild-type homomeric Kir4.1 (four H-bonds) and Kir4.1(K67M) channels (no H-bonds) suggesting that the number of H-bonds in the tetrameric channel complex determines the gating kinetics. Furthermore, in heteromeric Kir4.1(K67M)/Kir5.1 channels, where the two remaining H-bonds are disrupted, we found that the gating kinetics were similar to Kir4.1(K67M) homomeric channels despite the fact that these two channels differ considerably in their PIP₂ affinities. This indicates that Kir channel PIP₂ affinity has little impact on either the PIP₂- or pH-gating kinetics.     

Evidence against functional heteromultimerization of the KATP channel subunits Kir6.1 and Kir6.2

K(ATP) channels consist of pore-forming potassium inward rectifier (Kir6.x) subunits and sulfonylurea receptors (SURs). Although Kir6.1 or Kir6.2 coassemble with different SUR isoforms to form heteromultimeric functional K(ATP) channels, it is not known whether Kir6.1 and Kir6.2 coassemble with each other. To define the molecular identity of K(ATP) channels, we used adenoviral gene transfer to express wild-type and dominant-negative constructs of Kir6.1 and Kir6.2 in a heterologous expression system (A549 cells) and in native cells (rabbit ventricular myocytes). Dominant-negative (DN) Kir6.2 gene transfer suppressed current through heterologously expressed SUR2A + Kir6.2 channels. Conversely, DN Kir6.1 suppressed SUR2B + Kir6.1 current but had no effect on coexpressed SUR2A + Kir6. 2. We next probed the ability of Kir6.1 and Kir6.2 to affect endogenous K(ATP) channels in adult rabbit ventricular myocytes, using adenoviral vectors to achieve efficient gene transfer. Infection with the DN Kir6.2 virus for 72 h suppressed pinacidil-inducible K(ATP) current density measured by whole-cell patch clamp. However, there was no effect of infection with the DN Kir6.1 on the K(ATP) current. Based on these functional assays, we conclude that Kir6.1 and Kir6.2 do not heteromultimerize with each other and that Kir6.2 is the sole K(ATP) pore-forming subunit in the surface membrane of heart cells.